Aim 1: Design peptides that enable triggered hydrogelation Accomplishments: We have designed and studied well over a hundred peptides to gain an understanding of how sequence modulates folding, assembly and resultant material properties. Much effort has been dedicated to understanding the role of distinct structural perturbations on gelation as outlined below. 1. Strand number and registry. We assessed the effect of strand number on self-assembly by preparing single strand peptides and three-stranded beta-sheets for comparison with the two-stranded hairpin. We found that single strand peptides self-assemble to form weak gels composed of fibrils having heterogenous morphologies. 2. Turn type. In proteins, turns are responsible for chain reversal, help define the twist of sheets, and in some cases, nucleate folding. We designed the high propensity type II' (-VDPPT-) turn in MAX1 to include a beta-branched valine at position i to enforce a trans proline bond at the following residue, a -DPLP- unit at the central i+1 and i+2 positions to adopt dihedral angles similar to type II' turn in proteins, and a threonine at the i+3 position to form a side-chain/main chain H-bond to the ith carbonyl to stabilize the turn. We examined the influence of turn type on folding, assembly and gel mechanical properties by replacing this turn with canonical four-residue beta-turns and five residue [type I+G1 beta-buldge] sequences found in the literature. We found that the inherent folding propensity of each turn influenced the rate of hairpin folding and assembly, and that each turn type was capable of reversing the chain direction where intended, resulting in well-defined fibrils of uniform diameter. Importantly, rheology showed that turn type also influenced hydrogel mechanical rigidity. Diproline motifs within the four residue turns, and a five-residue [type I+G1 beta-buldge] sequence (VPDGT) containing a single proline, offered the stiffest gels of those studied. We are currently deriving a biophysical model to explain the dependence of turn type on gel network stiffness. 3. Perturbations to the hydrophobic face of the hairpin. Thermally-induced folding and assembly of our hairpins is driven by the hydrophobic effect, which is temperature dependent. We studied how hydrophobicity and side-chain identity influences the temperature-dependent folding and assembly of the hairpin, as well as hydrogel rheological properties. 4. Perturbations to the hydrophilic face of the hairpin. We have systematically studied how residue composition of the hydrophilic face of the hairpin affects folding, assembly and material properties. 5. Ligation of peptide epitopes and other functional groups. The function of the gels can be enhanced by covalently incorporating peptide epitopes and other chemistries into the fibrillar network via modification of the self-assembling hairpin. In general, the hairpin scaffold is forgiving of alterations to its sequence. Moieties can be incorporated at its N- and C-termini, as well as from its hydrophilic face by functionalizing the lysine side chains or incorporating non-natural residues; modifications at these regions minimally effect folding, assembly and bulk material properties. Changes to the hairpin's hydrophobic face are less forgiving. To date, we have incorporated various cell-binding epitopes (RGD, etc...) and peptide fragments capable of directing biomineralization. Smaller organic functionalities can also be incorporated, such as sorbamide groups from lysine side chains that allow photopolmerization of the fibrillar network. Taken together, our fundamental studies exploring peptide sequence-material relationships establish a continuously evolving basis set of design rules that allow us to rationally design peptides for targeted applications as will be shown throughout this report. Aim 2: Characterize hairpin folding, self-assembly mechanism, and resulting network structure. Accomplishments 1. Mechanistic understanding. Two mechanistic models for gelation were found that differ in their early steps. Initially, we favored mechanism A based on the literature describing hairpin folding, as well as our own data. However, recent publications describing the oligomerization of intrinsically disordered proteins, suggest that these early steps may be more complex as described in mechanism B, which we will investigate in future work (vide infra). 2. Network characteristics. Bulk rheological measurements of the final network indicate that our hydrogels display viscoeleastic behavior reminiscent of heavily crosslinked, semiflexible polymer networks such as actin whose physical properties can be predicted using Mackintosh theory. Aim 3: Study the encapsulation and delivery of small molecules, proteins, and cells. Accomplishments Our efforts to design gels for the local delivery of therapeutics have centered on proteins and cells. We have also recently started working towards small molecule delivery. 1. Delivery of proteins. We have shown that macromolecules can be directly and easily encapsulated in the gel network by adding a solution of unfolded peptide in water to a buffered solution of protein and triggering gelation. 2. Delivery of cells. Our work has focused on understanding how gel characteristics influence the encapsulation, delivery and behavior of cells for eventual use in tissue engineering and cytomedical therapy. 3. Small Molecule Delivery. We have recently begun exploring the potential of our gels to deliver small molecules. In collaboration, we showed that the small molecule, curcumin, could be encapsulated at therapeutically relevant concentrations in MAX8 gels without significantly influencing gel rheological properties. This hydrophobic compound is sparingly soluble in water, but can partition into the hydrophobic regions of the fibril assembly. We showed that curcumin can be released over days to effect action on model medulloblastoma cells. This study provided the impetus to propose the systematic studies described below that will establish the rules by which small molecules behave in, and are released from, the network. Aim 4: Develop hydrogels for Interleukin 7 (IL-7) delivery to modulate T cell survival. In healthy immune systems, consistent populations of peripheral lymphocytes are maintained through cytokines responsible for cell differentiation and proliferation. This is a new project and we have recently established two collaborations to help carry out the aims. Aim 5: Develop hydrogels that facilitate vascular anastomoses. This is a new collaborative project with Dr. Brandacher at Johns Hopkins, Department of Plastic Surgery. He and his team are leading experts in whole hand transplantation. We are developing gels that facilitate micro-vascular anastomoses, the suturing of very small vessels (Diameter 0.2mm) to aid organ transplantation. Accomplishments The Brandacher lab has developed a super-micro surgical model to study immunomodulatory effects in rat hind limb allotransplantation. This model can be adapted to study the efficacy of our gels in aiding anastomosis.